In time-of-flight mass spectrometers in which the samples are ionized by matrix-assisted laser desorption (MALDI), the laser beam is usually focused by fixed lenses and mirrors onto a sample on a sample support so that an irradiation spot with desired diameter and energy density is produced at a location in the acceleration system of the ion source which is optimally selected for high sensitivity. The sample contains a thin layer of small crystals of the matrix substance in which a small quantity of analyte molecules is embedded. A light pulse from the laser, usually a UV laser, is used to generate a plasma cloud of sample material in which ions of the matrix and analyte molecules are produced. Modern embodiments of MALDI lasers (U.S. Pat. No. 7,235,781) produce not just a single irradiation spot, but a pattern of several irradiation spots simultaneously, whereby the spot diameter and energy density can be optimized in such a way that a hundred times higher yield of analyte ions is achieved. The pattern can contain 4, 9 or 16 irradiation spots in a square arrangement, for example, but also 7 or 19 spots in a hexagonal arrangement. The utilization factor of the samples can be increased by using the sample material more economically. If a different spot on the sample has to be irradiated, the sample has to be moved by a movement of the sample support plate.
A voltage applied to diaphragms in the ion source accelerates the ions into a field-free flight tube. Since the ions have different masses, they are accelerated to different speeds in the ion source. Light ions reach the ion detector earlier than heavier ones. The ion currents are measured and digitized at the ion detector with two to eight measurements per nanosecond. The flight times of the ions are determined from the measured ion current values, and the masses of the ions are determined from the flight times. As is known to those skilled in the art, velocity-focusing reflectors can be used to increase the time-of-flight resolution. In particular, a delayed acceleration of the ions (DE=delayed extraction) can focus ions of one mass efficiently despite the initially broad distribution of their starting velocities brought about by the expanding plasma cloud. Summing 50 to 1,000 individual time-of-flight spectra from a sample to form a sum time-of-flight spectrum, and obtaining the mass spectrum of the sample is well-known Prior Art. Nowadays, mass resolutions of R=m/Δm>50,000 are achieved with good time-of-flight mass spectrometers, in a wide mass range of 1000 Da<m/z<4000 Da. The mass accuracies achieve values of the order of one millionth of the mass (1 ppm).
U.S. Pat. No. 6,734,421 discloses the synchronous acquisition of several mass spectra from several sample locations on a sample support, but without representing an explicit embodiment, is to introduce a beam deflection device for positioning the laser spot on the sample support plate. In the document it is proposed that the beam deflection could work with movable mirrors; piezo-controlled mirrors are expressly mentioned. The sample support should remain stationary while several samples are scanned; the ions of the different sample locations should be imaged onto different detectors. This should make it possible to measure mass spectra of several samples with a temporal overlap. In the document U.S. Published Patent Application 2004/0183009, mirrors are again used for positional control; here they are used to scan inhomogeneously prepared samples in order to find spots with higher ion yield (“sweet spots”). Also in U.S. Published Patent Application 2005/0236564 A1 a rotatable mirror is used to control the position of the laser spot in a direction vertical to the mechanical movement to generate a scanning raster. These solutions, however, do not consider fast spot control, using lasers with repetition rates up to 10 kilohertz, and moving the laser spot in the time span within two laser shots. In all these documents, relatively large mirrors were positioned near the optical lens system focusing the beam onto the sample. These mirrors have a relatively high inertia and cannot be redirected within 100 microseconds. Commercially available time-of-flight mass spectrometers with positional control for laser spots have not yet been developed.
Over the years, the laser technology for MALDI time-of-flight mass spectrometers has improved enormously. Not only has the division into several laser spots been introduced and become widespread under the name of “smartbeam”; the laser shot frequency has been constantly increased from initially 20 shots per second with nitrogen UV lasers to today's 1,000 to 5,000 shots per second with solid state UV lasers. The current goal is a repetition rate of 10 kHz, which means that only 100 microseconds are available for the acquisition of a time-of-flight spectrum, and also for the positional changes of the laser spots. With five ion current measurements per nanosecond at the detector, a single time-of-flight spectrum then consists of 500,000 measured values, enough for a mass resolution in the order of 50,000, and a mass accuracy of one part per million. As has already been mentioned, at least 50 to 1,000 individual time-of-flight spectra, which are added together at every mass position to fowl a sum time-of-flight spectrum, are acquired on one sample. This is then used to obtain the mass spectrum of the sample.
This technique with high laser shot rates is used especially in “imaging mass spectrometry” of thin tissue sections, with which many ten to hundred thousands of mass spectra are acquired from one thin tissue section. Just as an original color image contains a full color spectrum in each pixel, so a mass spectrometric image contains a full mass spectrum in each pixel. Pixel separations from 50 down to 20 micrometers are being used today, and the aim for the future is a spatial resolution of 10 or even 5 micrometers. 40,000 mass spectra are obtained from one square centimeter of thin tissue section at 50-micrometer resolution; at 10-micrometer resolution it is already a million mass spectra. In this case also, for the mass spectrum of one pixel, the individual flight-time spectra from 50 to 1,000 laser shots are added together to form a time-of-flight sum spectrum, from which the mass spectrum of the pixel is then obtained. The larger the number of individual time-of-flight spectra added together in each case, the better will be the detection limit and the signal-to-noise ratio. However, it is not always possible to acquire and add together any number of individual time-of-flight spectra because the sample is usually quickly exhausted.
The current state of the art is that these mass spectra are acquired with the laser spot or the laser spot pattern having a fixed position relative to the axis of the ion source. The spatial resolution is produced solely by the movement of the sample support plate. The required flatness of the surface means that the sample supports are quite bulky, with high inertia when taken together with the holder. The stepping movement of the sample support plate from sample site to sample site thus results in an extraordinarily high load for the movement device, which in general consists of a stepper motor and a threaded rod. At present, the sample support is moved with up to 10 sample sites per second; with 10 kHz lasers of the future it will have to be up to 200 sample sites per second and more, a movement which can no longer be achieved mechanically. As a solution for imaging mass spectrometry, attempts are already being made to work with a continuous movement of the sample support plate through a fixed laser spot position. This achieves a compromise between speed of forward movement and laser shot frequency in order to obtain a reasonably useful signal quality; moreover, the utilization of the sample is greatly limited. This operating mode, however, is not satisfactory for imaging mass spectrometry [see J. M. Spraggins and R. M. Caprioli, J. Am. Soc. Mass Spectrom. (2011) 22:1022-1031].
Moreover, uniform utilization of the available surface of a sample site, and thus utilization of the available analyte molecules for the acquisition of the individual time-of-flight spectra, is not very satisfactory at present. For example, nowadays, thin tissue sections for ionization by matrix-assisted laser desorption (MALDI) are prepared by applying a layer of tiny crystals of matrix material to the thin section; the soluble peptides and proteins are transported from the thin section into the top layer of the crystals. If the spot pattern is not moved, the analyte molecules under the laser spots are exhausted after three to five laser shots. Therefore, nowadays, the spot pattern is rotated with a swaying motion in order to repeatedly ablate as yet unused sites. To date, however, it is only possible to achieve really uniform ablation of a specified sample surface by moving the sample support plate. But the high frequency required for these movements is impossible to achieve today with the movement device for the sample support plate.
There is a need for a device for moving the sample support from fast, intermittent movements, both for the analysis of samples in high spatial density, as in imaging mass spectrometry, for example, and for the uniform ablation of the samples on specified areas.